Absolute quantification of particle number concentration using a digital single particle counting system.

Area of Science:

• Microfluidics
• Particle analysis
• Biotechnology

Background:

• Accurate particle concentration determination is crucial but challenging due to particle diversity.
• Existing methods often require difficult-to-obtain standard samples (calibrators).
• Single particle counting is essential for precise quantification, especially with heterogeneous particle populations.

Purpose of the Study:

• To develop a calibrator-free method for accurate single particle counting.
• To quantify particle concentrations over a broad dynamic range using microdroplet technology.
• To enhance signal-to-background ratio for improved digital counting of fluorescent particles.

Main Methods:

• Utilizing a microchip for digital counting and microdroplet formation.
• Compartmentalizing particles into picoliter droplets for individual counting.
• Applying Poisson statistics to calculate particle concentrations from positive droplet counts.
• Employing a T-junction microdroplet chip to increase signal-to-background ratio.

Main Results:

• Accurate determination of particle concentrations from 5.0 × 10^3 to 1.8 × 10^7 particles/mL without calibrators.
• A 9-fold increase in signal-to-background ratio achieved with a T-junction microdroplet chip compared to flow-focusing chips.
• Successful quantification of various fluorescent microspheres and heterogeneous bacteria (e.g., Escherichia coli DH5α-pDsRed).

Conclusions:

• The developed digital single particle counting system offers a widely applicable tool for absolute particle quantification.
• Microdroplet compartmentalization and digital counting provide accurate results for diverse and heterogeneous particles.
• This method eliminates the need for calibrators, simplifying particle quantification processes.